title: Alanine-scanning mutation approach for classification of the roles of conserved residues in the activity and substrate affinity of L-carnitine dehydrogenase.
abstract: L-Carnitine dehydrogenase (CDH) is as an excellent tool for L-carnitine (L-Car) estimation. To date, four CDHs have been identified, that share 45 % homology of their proteins. Here 42 conserved residues of CDH from Xanthomonas translucens (Xt-CDH) were substituted successively with alanine. The resultant mutants were analyzed for catalytic activity. Active mutants were evaluated for their influence on L-Car affinity. Twenty-three mutants with reduced affinity toward L-Car were subjected to detailed kinetic analysis. Analytical data implied that all mutants had increased K m values. The mutants of R193A, E196A, W199A, R200A, F249A, and F253A that produced the greatest L-Car affinity disruption (K m > 200-folds of Xt-CDH) clustered near the putative active site. This information can provide a solid basis for the rational design of mutagenic investigation to improve CDHs.
authors.affiliation: United Graduate School of Agricultural Sciences, Tottori University, Koyama, Tottori, 680-8553, Japan, mohamedmutasem@yahoo.com.
authors.forename: Mohamed M
authors.initials: MM
authors.lastname: Eltayeb
authors.forename: Jiro
authors.initials: J
authors.lastname: Arima
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Mori
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D002331:Carnitine / Q000378:metabolism*; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D007700:Kinetics; D014974:Xanthomonas / Q000201:enzymology* / Q000235:genetics
chemical_list: D000429:Alcohol Oxidoreductases; C019842:carnitine dehydrogenase; D002331:Carnitine